In eukaryotes, cytochrome c plays a vital role in both aerobic respiration and apoptosis, thus impacting the life and death of a cell. C-type cytochromes are characterized by covalent attachment of a heme co-factor, a modification that is required for its stability and subsequent function. Heme attachment takes place in the mitochondria and is mediated by holocytochrome c synthase, HCCS, the primary component of the eukaryotic cytochrome c biogenesis pathway, also known as System III. Previous studies in animals have shown that defects in HCCS can result in lethality or the human disease microphthalmia with linear skin defects (MLS). Although HCCS was discovered in yeast 25 years ago, the mechanisms underlying HCCS function have yet to be explored, largely due to its poor recombinant expression and instability. Our lab has very recently been successful in overcoming these technical limitations, allowing us to initiate the first comprehensive biochemical analysis of HCCS structure/ function. The catalytic function of HCCS depends on its ability to interact with and coordinate interactions between its substrates, heme and cytochrome c. Therefore, the proposed study seeks to determine which residues and/or domains in HCCS comprise its active site. Aim 1 analyzes the functional consequences of mutations of highly conserved residues in HCCS by examining perturbations in heme binding and cytochrome c recruitment and maturation. The apocytochrome c substrate-binding site of HCCS is directly assessed in Aim 2 by peptide crosslinking, using a UV-crosslinkable cytochrome c peptide containing the heme-attachment site. These studies will provide the first in-depth mechanistic analysis of HCCS, thus advancing our understanding of mitochondrial bioenergetics. Our results will directly impact what is currently known about diseases like MLS that are caused by HCCS abnormalities, as well as contribute to the body of knowledge concerning the many malfunctions observed in mitochondria in other human conditions.